Mass spectrometry of metallothionein adducts as candidate biomarkers of styrene oxide and 1-phenylpropylene oxide

Abstract

Metallothioneins (MT) are cysteine-rich intracellular proteins that sequester epoxides, such as the genotoxic styrene metabolite, styrene oxide (SO). Covalent adduct formation by the ring-opening of SO and its homologue, 1-phenylpropylene oxide (trans), with MT forms stable complexes that potentially may serve as biomarkers for epoxide exposure. MT was reacted with SO and trans-phenylpropylene oxide (PPO) separately in water. Mass spectra of adducts were obtained using electrospray ionization with positive-ion detection in an ion trap mass spectrometer. MS/MS analysis established the covalent nature of the complexation. Further experiments were conducted to determine the site of adduct formation. Molecular modeling was used to predict the most likely site of adduct formation. Alkylation and digestion were used to locate the peptide fragment where adduct formation occurred and confirm modeling results. The likeliest cysteine residue was residue 48 in the alpha domain of MT. MT-SO adducts provide a basis for the development of biomarkers. iii ACKNOWLEDGMENTS I would first like to thank my advisor, Dr. Patrick Callery, for his advice and confidence in me. He has been tremendous help to me in this whole process. I would also like to thank Dr. Grazyna Szklarz and Dr. Fred King for agreeing to serve on my committee. I want to also thank Dr. Diaa Shakleya for all of his help and guidance throughout this project. I want to also thank the entire Pharmaceutical Sciences Department, faculty, staff, and students for all of their help and support. I wish to thank Mylan Pharmaceuticals and my colleagues in the Bioanalytical Department, especially Dr. Scott Chervenick, who gave me the time and encouragement to continue my education. Finally, I would also like to especially thank my mother, family, and friends for all of their love, encouragement, and support. iv TABLE OF CONTENTS Abstract ii Acknowledgments iii Table of Contents iv List of Figures v List of Tables vi List of abbreviations vii